The activities of 17-beta- and 20-alpha-hydroxysteroid dehydrogenase in homogenates of human endometrium were measured using estradiol (E2), testosterone (T), 5-androstene-3beta, 17beta-diol (delta5-Adiol) and 20alpha-dihydroprogesterone (20alpha-DHP) as substrates. All of these activities were about 20 times higher in secretory tissue than in proliferative endometrium. The rates of oxidation of the three 17beta-hydroxysteriods tested were similar and about 4-5 times higher than the rate of oxidation of 20alpha-DHP, under the same assay conditions. Fragments of proliferative endometrium were incubated under organ culture conditions for 1 to 3 days either in medium alone or in medium to which various amounts of progesterone or medroxyprogesterone acetate were added. As reported before, 3-10 fold increases in 17beta-hydroxysteriod dehydrogenase activity, determined by measuring the rate of conversion of E2 to estrone, were noted during incubations in the presence of presence of progestins. Similar increases were observed when T, delta5-Adiol, and 20alpha-DHP were used as substrates in the enzymatic assay. The parallelism in response of these activities to progestin stimulation in vitro was also evident when time of incubation and concentration or type of progestins were varied. These results show that progestins enhance the activity of dehydrogenase involved in the metabolism of estrogens, androgens and progestins in human endometrium and provide evidence suggesting that these activities reside in a single dehydrogenase.